DNA不同提取方法对养殖池塘底泥细菌多样性PCR-DGGE检测的影响

Influence of Different DNA Extraction Methods on Microbial Diversity PCR-DGGE Detection of Culture Pond Sendiment

应用PCR-DGGE技术研究了养殖池塘底泥中细菌DNA 5种提取方法对细菌多样性检测的影响。结果显示:(1)DNA粗提液中腐殖酸与蛋白的纯度无正相关;(2)5种方法提取的DNA粗提液经过纯化后进行PCR均取得良好结果;(3)采用复合破壁方法提取养殖池塘底泥细菌DNA有利于显示细菌多样性;(4)DNA提取方法是影响池塘底泥细菌多样性PCR-DGGE结果的关键因素,与DNA的产量、纯度关系甚微;(5)5种方法相似性高达0.79,在对养殖池塘底泥细菌多样性进行粗略分析时均可使用;(6)本试验中,基于改进的Martin-Laurent法是用于养殖池塘底泥细菌多样性PCR-DGGE研究的较好的总DNA提取方法。

In this research,PCR-DGGE technique was used for studying five different extraction methods of microbial DNA of the pond sediment in difference of microbial diversities.The purpose is to try to find extraction methods of DNA which can be best used to objectively reflect the diversities and advantages of the bacteria of the pond sediment,and to evaluate comprehensively the five extraction methods.The results showed that： DNA in the crude extract of two humic acids and protein purity is of no positive correlation;the crude DNA extract of the five extraction methods,after purification,yields good results with PCR;broken composite method is used in the extraction of microbial DNA in the sediment ponds,helping enhance microbial diversity;DNA extraction method is the key factor of pond sediment microbial diversity by DGGE,and the diversity has poor relationship with the output and size of DNA;the similarity of the five ways is as high as 0.79,so it can be substituted for one another to conduct a rough analysis of microbial diversity;the improved Martin-Laurent Act is a better DNA extraction method for PCR-DGGE microbial diversity study in sediment ponds.